Synthesis and composition of amino acid linking groups conjugated to compounds used for the targeted imaging of tumors

ABSTRACT

The present disclosure relates to compounds that are useful as near-infrared fluorescence probes, wherein the compounds include i) a pteroyl ligand that binds to a target receptor protein, ii) a dye molecule, and iii) a linker molecule that comprises an amino acid or derivative thereof. The disclosure further describes methods and compositions for incorporating the compounds as used for the targeted imaging of tumors. Conjugation of the amino acid linking groups increase specificity and detection of the compound. Methods and compositions for use thereof in diagnostic imaging are contemplated.

RELATED APPLICATIONS

The present patent application is a continuation of U.S. patentapplication Ser. No. 15/584,752, filed May 2, 2017, which is acontinuation-in-part of U.S. patent application Ser. No. 15/259,719,filed Sep. 8, 2016, now U.S. Pat. No. 9,789,208, which is acontinuation-in-part of U.S. patent application Ser. No. 14/953,928,filed Nov. 30, 2015, now U.S. Pat. No. 9,782,497, which is acontinuation of U.S. patent application Ser. No. 14/715,799, filed May19, 2015, now U.S. Pat. No. 9,341,629, which is a continuation of U.S.patent application Ser. No. 14/207,130, filed Mar. 12, 2014, now U.S.Pat. No. 9,061,057, which is a continuation of U.S. patent applicationSer. No. 14/010,098, filed Aug. 26, 2013, now U.S. Pat. No. 9,333,270,which is related to and claims priority benefit of U.S. ProvisionalPatent Application No. 61/791,921, filed Mar. 15, 2013, all of which arehereby incorporated by reference in their entirety into this disclosure.

FIELD OF THE DISCLOSURE

The present disclosure is in the area of diagnostics. This disclosureprovides methods of synthesizing and utilizing amino acid linking groupsthat are conjugated to a compound used for the targeted imaging oftumors. Conjugation of the amino acid linking groups increasespecificity and detection of the compound. Methods and compositions foruse thereof in diagnostic imaging are contemplated.

BACKGROUND OF THE DISCLOSURE

Surgical removal of malignant disease constitutes one of the most commonand effective therapeutic for primary treatment for cancer. Resection ofall detectable malignant lesions results in no detectable return of thedisease in approximately 50% of all cancer patients' and may extend lifeexpectancy or reduce morbidity for patients in whom recurrence of thecancer is seen. Not surprisingly, surgical methods for achieving morequantitative cytoreduction are now receiving greater scrutiny.

Resection of all detectable malignant lesions results in no detectablereturn of the disease in approximately 50% of all cancer patients andmay extend life expectancy or reduce morbidity for patients in whomrecurrence of the cancer is seen. Given the importance of totalresection of the malignant lesions, it is beneficial to ensure that themalignant lesions are accurately and completely identified.Identification of malignant tissue during surgery is currentlyaccomplished by three methods. First, many tumor masses and nodules canbe visually detected based on abnormal color, texture, and/ormorphology. Thus, a tumor mass may exhibit variegated color, appearasymmetric with an irregular border, or protrude from the contours ofthe healthy organ. A malignant mass may also be recognized tactilely dueto differences in plasticity, elasticity or solidity from adjacenthealthy tissues. Finally, a few cancer foci can be locatedintraoperatively using fluorescent dyes that flow passively from theprimary tumor into draining lymph nodes. In this latter methodology,fluorescent (sentinel) lymph nodes can be visually identified, resectedand examined to determine whether cancer cells have metastasized tothese lymph nodes.

Despite the recognition of the importance of removal of tumor and theavailability of certain identification techniques for visualizing tumormass, many malignant nodules still escape detection, leading to diseaserecurrence and often death. Thus, there is a need for improved tumoridentification. This motivation has led to introduction of two newapproaches for intraoperative visualization of malignant disease. In thefirst, a quenched fluorescent dye is injected systemically into thetumor-bearing animal, and release of the quenching moiety by atumor-specific enzyme, pH change, or change in redox potential isexploited to selectively activate fluorescence within the malignantmass. In the second approach, a fluorescent dye is conjugated to atumor-specific targeting ligand that causes the attached dye toaccumulate in cancers that over-express the ligand's receptor. Examplesof tumor targeting ligands used for this latter purpose include folicacid, which exhibits specificity for folate receptor (FR) positivecancers of the ovary, kidney, lung, endometrium, breast, and colon, andDUPA, which can deliver attached fluorescent dyes selectively to cellsexpressing prostate-specific membrane antigen (PSMA), i.e. prostatecancers and the neovasculature of other solid tumors. Beneficially, onefolate-targeted fluorescent dye (folate-fluorescein or EC17) has beenrecently tested intra-operatively in human ovarian cancer patients. Inthis study, ˜5× more malignant lesions were removed with the aid of thetumor-targeted fluorescent dye than without it, and all resectedfluorescent lesions were confirmed by pathology to be malignant.

Conventional fluorescent techniques use probes in the visible lightspectrum (˜400-600 nm), which is not optimal for intra-operativeimage-guided surgery as it is associated with a relatively high level ofnonspecific background light due to collagen in the tissues. Hence thesignal to noise ratio from these conventional compounds is low.Moreover, the absorption of visible light by biological chromophores, inparticular hemoglobin, limits the penetration depth to a fewmillimeters. Thus tumors that are buried deeper than a few millimetersin the tissue may remain undetected. Moreover ionization equilibrium offluorescein (pKa=6.4) leads to pH-dependent absorption and emission overthe range of 5 to 9. Therefore, the fluorescence of fluorescein-baseddyes is quenched at low pH (below pH 5).

For example, the potential use of EC17 dye for a more widespread use inoptical imaging for the characterization and measurement diseased tissuein a clinical setting has been hampered by the major drawback of thatthe attached dye (fluorescein) emits fluorescence in the visible range.This makes EC17 and related dyes poor for in vivo use in tissues becausetissues typically autofluoresce strongly in the visible range, and lightpenetrates tissue poorly. Moreover, EC17(folate-ethelenediamine-fluorescein isothiocynate) consists a thiourealinker. It is well known that thiourea compounds have low shelf life dueto the instability of the thiourea linkage. Thus, a compound such asEC17 is not optimal for use in optical imaging because of thisunstability and the related decomposition of the decomposition ofthiourea bridge.

The combination of light absorption by hemoglobin in the visible lightspectrum (<600 nm) and water and lipids in the IR range (>900 nm),offers an optical imaging window from approximately 650-900 nm in whichthe absorption coefficient of tissue is at a minimum. A suitablealternative to dyes that emit light in the visible range would be todevelop dyes that can be used in the near infra-red (NIR) range becauselight in the near infrared region induces very little autofluorescenceand permeates tissue much more efficiently. Another benefit to near-IRfluorescent technology is that the background from the scattered lightfrom the excitation source is greatly reduced since the scatteringintensity is proportional to the inverse fourth power of the wavelength.Low background fluorescence is necessary for highly sensitive detection.Furthermore, the optically transparent window in the near-IR region (650nm to 900 nm) in biological tissue makes NIR fluorescence a valuabletechnology for in vivo imaging and subcellular detection applicationsthat require the transmission of light through biological components.

While the use of light in the NIR range for deeper tissue imaging ispreferable to light in the visible spectrum, the NIR imaging dyescurrently used in the art suffer from a number of challenges anddisadvantages such as a susceptibility to photobleach, poor chemicalstability, absorbance and emission spectra that fall within the samerange as many physiological molecules (resulting in high backgroundsignal and autofluorescence). Moreover, most of the NIR dyes are notstable during the synthesis, especially conjugating to a ligand with anamine linker, leading to multiple unwanted side products. Therefore,taking ligand-targeted NIR imaging agent for clinic can be expensive.Thus, current imaging methods that utilize NIR fluorescent probes arenot effective in deep tissue imaging (>5 mm from the surface), inquantifying fluorescence signal in mammalian tissues, or in productioncost that increase preclinical-to-clinical translational time.

Two promising approaches to fluorescence-guided surgery are currentlyunder intense investigation for use in the clinic. In one method, anactivatable NIR fluorescent probe, which is minimally fluorescent in thesteady state due to its proximity to an attached quencher, becomeshighly fluorescent upon release of the quencher in malignant tissue. Oneof the most commonly used release mechanisms involves incorporation of apeptide sequence between the dye and the quencher that can bespecifically cleaved by a tumor-enriched protease (i.e. cathepsins,caspases and matrix metalloproteinases). A major advantage of thisstrategy lies in the absence of fluorescence in tissues that lack theactivating enzyme, allowing tissues along the excretion pathway (e.g.kidneys, bladder, liver) to remain nonfluorescent unless theyfortuitously express the cleaving enzyme. Such tumor-activated NIR dyescan also generate substantial fluorescence in the tumor mass as long asthe malignant lesion is enriched in the cleaving protease and thereleased dye is retained in the tumor. The major disadvantage of thismethodology arises from the poor tumor specificities of many of therelevant hydrolases (most of which are also expressed in healthy tissuesundergoing natural remodeling or experiencing inflammation). Moreover,the abundance of the desired proteases may vary among tumor masses,leading to slow or no activation of fluorescence in some malignantlesions and rapid development of fluorescence in others.

Thus, there remains a need for a dye substance that can be used tospecifically target diseased tissue and has increased stability andbrightness for use in vivo for tissue imaging.

BRIEF SUMMARY OF THE DISCLOSURE

This disclosure provides a method for synthesizing amino acid linkinggroups that are conjugated to a compound used for the targeted imagingof tumors and lymph nodes. In certain embodiments, this disclosurerelates to a compound or a salt derivative thereof, that comprises afolate or pteroyl ligand, a linking group, and a fluorescent dye. Incertain embodiments, the linking group can be an amino acid, an isomer,a derivative, or a racemic mixture thereof. In other aspects, thefluorescent dye is selected from the group consisting of LS288, IR800,SP054, S0121, KODAK, S2076 and S0456.

In some aspects, this disclosure provides a method of conjugating anamino acid linking group to a fluorescent dye, wherein the amino acidcan be tyrosine, serine, theronine, lysine, arginine, asparagine,glutamine, cysteine, selenocysteine, isomers, and the derivativesthereof. In certain embodiments, the amino acid, isomers, or thederivatives thereof, contain an —OH, —NH₂, or —SH functional group thatupon addition of the fluorescent dye in slight molar excess produces theconjugation of fluorescent group with the amino acid, isomer, or thederivatives thereof. In other embodiments, the amino acid, isomers, orthe derivatives thereof, contains an —OH functional group that uponsynthesis generates an ether bond with the fluorescent dye thatincreases the brightness and detection of the compound. In someembodiments, this disclosure relates to the conjugation of the aminoacid linking group with the fluorescent dye, wherein the amino acid,isomers, or the derivatives thereof, contains an —SH, —SeH, —PoH, or—TeH functional group that upon synthesis generates a C—S, C—Se, C—Po,or C—Te bond with the fluorescent dye. In some aspects, this disclosurerelates to the conjugation of the amino acid linking group to afluorescent dye that has an absorption and emission maxima between about500 nm and about 900 nm. In other aspects, the amino acid linking groupis conjugated to a fluorescent dye that has an absorption and emissionmaxima between about 600 nm and about 800 nm.

In additional embodiments, this disclosure provides a method forconjugating the amino acid linking group to a folate ligand, wherein theamino acid linking group is tyrosine, serine, theronine, lysine,arginine, asparagine, glutamine, cysteine, selenocysteine, isomers orthe derivatives thereof, and is conjugated to folate through a dipeptidebond. In additional aspects, this disclosure provides a method ofconjugating the linking group with a folate ligand, wherein the linkinggroup is tyrosine, serine, theronine, lysine, arginine, asparagine,glutamine, cysteine, selenocysteine, isomers, or the derivativesthereof, and is conjugated to folate through a homo-oligopeptide bond.In other embodiments, this disclosure relates to a method of conjugatinga pteroyl ligand to an amino acid linking group, wherein the linkinggroup is tyrosine, serine, theronine, lysine, arginine, asparagine,glutamine, cysteine, selenocysteine, isomers or the derivatives thereof.In certain aspects, the carboxylic acid of the linking group is bound tothe alpha carbon of any amino acid, hence increasing the specificity ofthe compound for targeted receptors. In some embodiments, the amino acidlinking group contributes specificity to the compound, wherein theobserved binding affinity of the compound to targeted receptors isfolate receptor.

In additional aspects, the compound is highly selective for targeting totumor cells expressing the target receptor.

In other embodiments, this disclosure relates to the use of a compounddesignated, Pte-Tyr-S0456 (OTL-0038) for image guided surgery, tumorimaging, lymph node imaging, inflammatory diseases, atherosclerosis,infection diseases, forensic applications, mineral applications, dental,gel staining, DNA sequencing, nerve staining, or plastic surgery. Inother aspects, the Pte-Tyr-S0456 derivative can be Pte-D-Tyr-S0456,Pte-homoTyr-S0456, Pte-beta-homo-Tyr-S0456, Pte-(NMe)-Tyr-S0456,Pte-Tyr(OMe)-S0456, Pte-Tyr(OBn)-S0456, Pte-NHNH-Tyr-OAc-S0456, salts,or derivatives thereof.

In other aspects, this disclosure provides a method of synthesizing thecompound, wherein a protecting group is used to avoid undesiredreactivity with groups other than the amino groups that might generateunwanted compounds. The methods provided in this disclosure produce afinal compound with a yield of over 98% purity.

In certain aspects, this disclosure relates to a compound used for thetargeted imaging of tumors, wherein the compound could be used forresearch, diagnostic, or therapeutic purposes. In other embodiments,this disclosure provides a composition comprising an imaging compoundand a pharmaceutically acceptable carrier, excipient, diluents, orsalts.

-   -   wherein W, X, Y, or Z is H, Na, or NH₄ ⁺,

-   -   wherein W, X, Y, or Z is H, Na, or NH₄ ⁺,

-   -   wherein W, X, Y, or Z is H, Na, or NH₄ ⁺,

BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 depicts the design of OTL38.

FIG. 2 depicts the chemical structures, excitation and emission spectraof OTL38.

FIG. 3A depicts binding and internalization of OTL38 to human cervicalcancer (KB-FR⁺) cells by confocal microscopy.

FIG. 3B depicts binding and internalization of OTL38 to human cervicalcancer (KB-FR⁺) cells by confocal microscopy.

FIG. 3C depicts binding and internalization of OTL38 to human lungcancer (A549-FR⁻) cells by confocal microscopy.

FIG. 3D depicts binding and internalization of OTL38 to human breastcancer (MDA-MB 231-FR⁺) cells by confocal microscopy.

FIG. 3E depicts binding and internalization of OTL38 to human ovariancancer (SKOV3-FR⁺) cells by confocal microscopy.

FIG. 3F depicts binding and internalization of OTL38 to human lungcancer (A549-FR⁻) cells by bright field image from confocal microscopy.

FIG. 4A depicts an overlay of whole body fluorescence image over whitelight image (after adjusting the threshold). Mice were injected with 10nmol of OTL38, euthanized and imaged with IVIS imager 2 hours postinjection. Exposure time=1 s; Excitation: 745 nm. Emission: ICG(indocyanine green). Mice were bearing human cervical tumor xenografts.

FIG. 4B depicts tissue biodistribution analysis using fluorescenceimaging of two mice from FIG. 4A.

FIG. 4C depicts tumor-to-tissue ratio from tissue biodistribution dataof human cervical tumor xenograft model of FIG. 4A.

FIG. 5A depicts an overlay of whole body fluorescence image over whitelight image after adjusting the threshold. The mouse was injected with10 nmol of OTL38, euthanized and imaged with IVIS imager 24 hours postinjection and analyzed using ImageJ analysis software. Exposure time=1s; Excitation: 745 nm. Emission: ICG (indocyanine green). Mouse wasbearing human cervical tumor xenografts.

FIG. 5B depicts a gray scale fluorescence of the mouse of FIG. 6A.

FIG. 5C depicts a plot of gray value Vs distance (c) across the lineshown in FIG. 5B.

FIG. 5D depicts a gray scale fluorescence of the mouse of FIG. 5 a.

FIG. 5E depicts a 3D plot of gray value Vs within the box shown in FIG.5D for a mouse bearing human cervical tumor xenografts.

FIG. 6A depicts a tissue biodistribution analysis using fluorescenceimaging at 0.3 nmol dose of OTL38. Human cervical tumor xenograftsbearing mice were injected with 2 nmol of OTL38, euthanized and imagedwith IVIS imager 2 hours post injection. Exposure time=1 s; Excitation:745 nm. Emission: ICG (indocyanine green).

FIG. 6B depicts a tissue biodistribution analysis using fluorescenceimaging at 1.0 nmol dose of OTL38.

FIG. 6C depicts a tissue biodistribution analysis using fluorescenceimaging at 3.0 nmol dose of OTL38.

FIG. 6D depicts a tissue biodistribution analysis using fluorescenceimaging at 10 nmol dose of OTL38.

FIG. 6E depicts a tissue biodistribution analysis using fluorescenceimaging at 20 nmol dose of OTL38.

FIG. 6F depicts a tissue biodistribution analysis using fluorescenceimaging at 30 nmol dose of OTL38.

FIG. 6G depicts a tissue biodistribution analysis using fluorescenceimaging at 60 nmol dose of OTL38.

FIG. 6H depicts a tissue biodistribution analysis using fluorescenceimaging at 90 nmol dose of OTL38.

FIG. 6I depict plots of a tissue biodistribution analysis usingfluorescence imaging at FIGS. 6A-HC dose levels of OTL38.

FIG. 7A depicts an overlay of whole body fluorescence image over whitelight images (after adjusting the threshold). Human cervical tumorxenograft bearing mice injected with 2 nmol of compound 4 and image withIVIS imager at 2 hours post injection. Exposure time=1 s; Excitation:745 nm. Emission: ICG (indocyanine green).

FIG. 7B depicts a tissue biodistribution analysis using fluorescenceimaging of two mice of FIG. 7A.

FIG. 7C depicts to umor-to-tissue ratio from tissue biodistribution dataof a human cervical tumor xenograft bearing mice of FIG. 7A.

FIG. 8 depicts an overlay of time dependent whole body fluorescenceimage over white light images before adjusting the threshold. Humancervical tumor bearing mice were injected with 10 nmol of OTL38 andimage with IVIS imager at different time intervals. Exposure time=1 s;Excitation: 745 nm. Emission: ICG (indocyanine green).

FIG. 9A depicts an overlay of whole body fluorescence image over whitelight images (after adjusting the threshold). Human ovarian (IGROV)xenograft tumor bearing mice injected with 2 nmol of OTL38 and imagewith IVIS imager at 2 hours post injection. Exposure time=1 s;Excitation: 745 nm. Emission: ICG (indocyanine green).

FIG. 9B depicts a tissue biodistribution analysis using fluorescenceimaging of mice of FIG. 9A.

FIG. 9C depicts a tumor-to-tissue ratio from tissue biodistribution dataof mice of FIG. 9A.

FIG. 10A depicts a tissue biodistribution analysis as an overlay offluorescence image over white light image (after adjusting thethreshold) of selected tissues of a human ovarian (IGROV) orthotropictumor mouse injected with 2 nmol of OTL38 and imaged with IVIS imager at2 hours post injection. Exposure time=1 s; Excitation: 745 nm. Emission:ICG (indocyanine green).

FIG. 10B depicts a tissue biodistribution analysis as tumor-to-tissueratio from mouse of FIG. 10A.

FIG. 11A depicts an overlay of half body fluorescence image over whitelight image (after adjusting the threshold). Tissue biodistribution dataof a human ovarian (SKOV3) tumor bearing mice injected with 2 nmol ofOTL38 and image with IVIS imager at 2 hours post injection. Exposuretime=1 s; Excitation: 745 nm. Emission: ICG (indocyanine green).

FIG. 11B depicts a tissue biodistribution analysis using fluorescenceimaging of mice of FIG. 11A.

FIG. 11C depicts a tumor-to-tissue ratio from tissue biodistributiondata of mice of FIG. 11C.

FIG. 12A depicts a tissue biodistribution analysis of overlay offluorescence image over white light image (after adjusting thethreshold) of selected tissues. Ovarian (SKOV3) orthotopic tissuebiodistribution data of mouse injected with 2 nmol of OTL38 and imagewith IVIS imager at 2 hours post injection. Exposure time=1 s;Excitation: 745 nm. Emission: ICG (indocyanine green). Kidneys wereremoved from the biodistribution images.

FIG. 12B depicts an image of ovarian orthotopic ovaries of mouse of FIG.12A.

FIG. 12C depicts a tumor-to-tissue ratio from tissue biodistributiondata of mouse of FIG. 12A.

FIG. 13A depicts an overlay of whole body fluorescence image over whitelight image (after adjusting the threshold). Tissue biodistribution dataof MDA-MB 231 xenograft breast cancer tumor bearing mice injected with 2nmol of OTL38 and image with IVIS imager at 2 hours post injection.Exposure time=1 s; Excitation: 745 nm. Emission: ICG (indocyaninegreen).

FIG. 13B depicts a tissue biodistribution analysis using fluorescenceimaging of mice of FIG. 13A.

FIG. 13C depicts a tumor-to-tissue ratio from tissue biodistributiondata of mice of FIG. 13A.

FIG. 14A depicts an overlay of whole body fluorescence image over whitelight image (after adjusting the threshold) of human breast tumorxenografts (MDA-MB 231). Tissue biodistribution data of human breast(MDA-MB 231) orthotopic tumor bearing mice injected with 2 nmol of OTL38and image with IVIS imager at 2 hours post injection. Exposure time=1 s;Excitation: 745 nm. Emission: ICG (indocyanine green).

FIG. 14B depicts a tissue biodistribution analysis using fluorescenceimaging of mice of FIG. 14A.

FIG. 14C depicts a tumor-to-tissue ratio from tissue biodistributiondata of mice of FIG. 14A.

FIG. 14D depicts human breast cancer cells metastasized to lung.

FIG. 15A depicts an overlay of whole body fluorescence image over whitelight image (after adjusting the threshold) a murine leukemia cellsmetastasized liver, lung and spleen. Tissue biodistribution data of amurine leukemia cancer (L1210A) metastatic tumor bearing mice injectedwith 2 nmol of OTL38 and image with IVIS imager at 2 hours postinjection. Exposure time=1 s; Excitation: 745 nm. Emission: ICG(indocyanine green).

FIG. 15B depicts an overlay of whole body fluorescence image over whitelight image (after adjusting the threshold) of mice of FIG. 15A.

FIG. 15C depicts a tumor-to-tissue ratio from tissue biodistributiondata of mice of FIG. 15A.

FIG. 16A depicts an overlay of whole body fluorescence image over whitelight image (after adjusting the threshold). Tissue biodistribution dataof a murine lung cancer (M109) metastatic tumor bearing mice injectedwith 2 nmol of OTL38 and image with IVIS imager at 2 hours postinjection. Exposure time=1 s; Excitation: 745 nm. Emission: ICG(indocyanine green).

FIG. 16B depicts a tissue biodistribution analysis using fluorescenceimaging of mice of FIG. 16A.

FIG. 17A depicts an overlay of whole body fluorescence image over whitelight image (after adjusting the threshold), 2 h after administering 2nmol of OTL38 into a mouse bearing a human breast tumor xenograft.Images were acquired with IVIS imager at 2 h post injection. Exposuretime=1 s; Excitation: 745 nm. Emission: ICG (indocyanine green).

FIG. 17B depicts an overlay of whole body fluorescence image over whitelight image (after adjusting the threshold), 2 h after administering 100fold excess of folic acid followed by 2 nmol of OTL38 into a mousebearing a human breast tumor xenograft. Images were acquired with IVISimager at 2 h post injection. Exposure time=1 s; Excitation: 745 nm.Emission: ICG (indocyanine green).

FIG. 17C depicts a tissue biodistribution of mice of FIG. 17A.

FIG. 17D depicts a tissue biodistribution of mice of FIG. 17B

FIG. 18A depicts an overlay of whole body fluorescence image over whitelight image (after adjusting the threshold) of a human lung tumorxenograft that do not express FR. Three mice from tissue biodistributiondata of A549 tumor bearing mice injected with 2 nmol of OTL38 and imagewith IVIS imager at 2 hours post injection. Exposure time=1 s;Excitation: 745 nm. Emission: ICG (indocyanine green).

FIG. 18B depicts a tissue biodistribution of mice of FIG. 18A.

DETAILED DESCRIPTION OF THE DISCLOSURE

Surgery is one of the best therapies for all the solid tumors, such asprostate, ovarian, lung, breast, colon, and pancreatic cancer. Whilesurgery is effective in 50% of patients with solid tumors in the US,chemo- and radiotherapy alone are effective in less than 5% of allcancer patients. Over 700,000 patients undergo cancer surgery every yearin the US and 40% of surgical patients have a recurrence of locoregionaldisease within 5 years. Despite major advances in the oncology fieldover the last decade, there remain significant hurdles to overcome inthe field. For example, it remains difficult to achieve completeresection of the primary tumor with negative margins, removal of thelymph nodes harboring metastatic cancer cells and identification ofsatellite disease. Achieving improvements in these three cases not onlyimproves disease clearance but also guides decisions regardingpostoperative chemotherapy and radiation. While non-targeted fluorescentdyes have been shown to passively accumulate in some tumors, theresulting tumor-to-background ratios are often poor and the boundariesbetween malignant and healthy tissues can be difficult to define.Although ligand targeted fluorescence dyes (e.g., EC17: Folate-EDA-FITC)have been used for imaging a tissue, those dyes have been ineffective asthey would not penetrate deep tissue and hence only identified thespecific cells on the surface of a tissue rather than deeper within thetissue sample. In addition, it has been shown that the excitation andemission spectra of these previous fluorescence dyes was such that itproduced significant background noise such that the targeted tissue wasnot easily detected. In addition, as discussed in the background above,fluorescein-based dyes have the disadvantages that of low shelf-lifestability. EC17 easily decomposes as a result of the instability of thethiourea bridge in that compound. In addition, as EC17 uses fluoresceinwhich has the drawback of a relatively high level of nonspecificbackground noise from collagen in the tissues surrounding the imagingsite. Moreover, the absorption of visible light by biologicalchromophores, in particular hemoglobin, further limits the usefulness ofdyes that incorporate fluorescein. This means that conventional dyescannot readily detect tumors that may be buried deeper than a fewmillimeters in the tissue. Furthermore, fluorescence from fluorescein isquenched at low pH (below pH 5)

In order for a dye material to be useful in detecting and guidingsurgery or providing other tissue imaging it is important to overcomethese drawbacks.

Several criteria were considered in preparation of conjugates includingnear infrared dyes. Ease of synthesis and chemical stability wereprimary chemical attributes. Spectral properties, such as absorption andemission spectra and quantum yield, were considered. Several biologicalproperties were evaluated, such as binding affinity in cell studies,whole body animal imaging using mice with tumors, and biodistribution.Specifically for biodistribution several aspects were consideredincluding dead mice after 2 hours per oral distribution, live miceimaging and dose escalation. Finally, safety considerations were takenincluding Maximum Tolerance Dose (MTD), ImmunoHistoChemical (IHC)analysis, and general clinical pathology analysis.

The present disclosure provides pteroyl conjugates of near infrared dyesthat are stable, fluoresce in the infrared range, and penetrate deepwithin targeted tissue to produce a specific and bright identificationof areas of tissue that express folate receptor. More specifically, thepteroyl conjugates are linked to the near infrared dyes through an aminoacid linker. Even more specifically, it has been found that where theamino acid linker is tyrosine or a derivative of tyrosine, the intensityof the fluorescence of the dye is maintained or even enhanced.

An amino acid is defined as including an amine functional group linkedto a carboxylic acid functional group, and a side-chain specific to eachamino acid. An alpha amino acid is any compound of the general formulaR⁵CH(NH₂)COOH (α-amino acid), wherein R⁵ is selected from the groupconsisting of H or any known amino acid side chain.

A beta amino acid is defined as including an amine functional grouplinked at a beta carbon and a carboxylic acid functional group linked atthe alpha carbon. A beta homo amino acid is defined as including anamine functional group linked at a beta carbon, a carboxylic acidfunctional group linked at the alpha carbon and a side-chain starting ateither the alpha carbon or the beta carbon wherein the side-chain isbound to another amino acid.

Naturally occurring amino acids can be divided into the following fourgroups: (1) acidic amino acids, (2) basic amino acids, (3) neutral polaramino acids, and (4) neutral nonpolar amino acids. Representative aminoacids within these various groups include, but are not limited to: (1)acidic (negatively charged) amino acids such as aspartic acid andglutamic acid; (2) basic (positively charged) amino acids such asarginine, histidine, and lysine; (3) neutral polar amino acids such asglycine, serine, threonine, cysteine, tyrosine, asparagine, andglutamine; and (4) neutral nonpolar (hydrophobic) amino acids such asalanine, leucine, isoleucine, valine, proline, phenylalanine,tryptophan, and methionine.

Conserved substitution for an amino acid within a naturally occurringamino acid sequence can be selected from other members of the group towhich the naturally occurring amino acid belongs. For example, thealiphatic side chains group of amino acids is glycine, alanine, valine,leucine, and isoleucine. Conserved substitution of naturally occurringamino acid valine includes use of glycine, alanine, leucine, orisoleucine.

The aliphatic-hydroxyl side chain group of amino acids is serine andthreonine. The amide-containing side chain group of amino acids isasparagine and glutamine. The aromatic side chain group of amino acidsis phenylalanine, tyrosine, and tryptophan. The basic side chain groupof amino acids is lysine, arginine, and histidine. The sulfur-containingside chain group of amino acids having is cysteine and methionine.Examples of naturally conservative amino acids substitutions are: valinefor leucine, serine for threonine, phenylalanine for tyrosine, lysinefor arginine, cysteine for methionine, and asparagine for glutamine.

In preferred embodiments, it is shown herein that such pteroylconjugates specifically target to tumor cells within a tissue. Moreover,the intensity of the fluorescence in greater than the intensity ofpreviously observed with other near infrared dyes that are targeted withfolate for folate receptor positive tumors. This increased intensityallows the targeting and clear identification of smaller areas ofbiological samples (e.g., smaller tumors) from a tissue being monitored.In addition, the increased intensity of the compounds of the presentdisclosure provides the added advantage that lower doses/quantities ofthe dye can be administered and still produces meaningful results. Thus,the compounds of the present disclosure lead to more economical imagingtechniques. Moreover, there is an added advantaged that a lower dose ofthe compounds of the disclosure as compared to conventional imagingcompounds minimizes the toxicity and other side effects that areattendant with administration of foreign materials to a body.

Furthermore, identification of small tumors will lead to a more accurateand more effective resection of the primary tumor to produce negativemargins, as well as accurate identification and removal of the lymphnodes harboring metastatic cancer cells and identification of satellitedisease. Each of these advantages positively correlates with a betterclinical outcome for the patient being treated.

In specific experiments, it was found that use of amino acids other thantyrosine as the linker resulted in loss of near infrared fluorescence.For example, see discussion of Scheme I. Specifically note the syntheticpathway lead to undesired by-product 4 as major product that does nothave NIR properties

However, it is contemplated that in addition to tyrosine and tyrosinederivatives, a pteroyl conjugate of a near infrared dye with cysteine orcysteine derivatives also may be useful. Furthermore, it is contemplatedthat a direct linkage of the pteroyl or folate moiety to the dye orlinkage of the dye to pteroic acid or folic acid through an amine linkeralso produces a loss of intensity of the fluorescence from the conjugatewhereas the presence of the tyrosine or tyrosine derivative as thelinking moiety between the pteroyl (targeting moiety) and the nearinfrared dye (the fluorescing moiety) is beneficial to maintain orenhance the fluorescence of the conjugated compound. Tyrosine-basedcompounds of the disclosure do not require an extra amine linker toconjugate the S0456 and further because conjugation through the phenolmoiety of the tyrosine leads to enhanced fluorescence.

The compounds can be used with fluorescence-mediated moleculartomographic imaging systems, such as those designed to detectnear-infrared fluorescence activation in deep tissues. The compoundsprovide molecular and tissue specificity, yield high fluorescencecontrast, brighter fluorescence signal, and reduce backgroundautofluorescence, allowing for improved early detection and moleculartarget assessment of diseased tissue in vivo (e.g., cancers). Thecompounds can be used for deep tissue three dimensional imaging,targeted surgery, and methods for quantifying the amount of a targetcell type in a biological sample.

Compounds

In an aspect the disclosure relates to compounds comprising the formula:Formula (I):

wherein:

X is an amino acid or a derivative thereof, and

Y is a dye that has a fluorescence excitation and emission spectra inthe near infra-red range, and said compound maintains or enhances thefluorescence of Y.

In some embodiments, the amino acid or amino acid derivative induces ashift in the electronic emission spectrum, the electronic absorptionspectrum, or both the electronic emission and absorption spectrum,relative to the electronic spectra of the unmodified dye molecule.Suitably, the shift in the electronic spectrum is a bathochromic shift(i.e., shift to longer wavelength/lower frequency) that helps to improvethe detection of the compound in the near infrared (NIR) spectral windowand/or reduce the amount of background signal, auto-fluorescence,interferences from the tissue surrounding the area being visualized.More specifically, this shift in electronic spectrum is particularlyobserved with NIR dyes that comprise electronegative atoms that areincorporated into the 6-membered ring. Thus, in certain embodiments theamino acid or amino acid (X) derivative comprises an electron-richmoiety such as, for example, oxygen, sulfur, or nitrogen. Non-limitingexamples of such amino acids can include cysteine, methionine,threonine, serine, tyrosine, phenylalanine, tryptophan, histidine,lysine, arginine, aspartic acid, glutamic acid, asparagine, andglutamine, or derivatives thereof.

In embodiments of this aspect, the disclosure provides compounds ofFormulas (I)a, (I)b, (I)c, and (I)d:

wherein the Tyr, Cys, Ser, and Lys groups indicate a tyrosine, acysteine, a serine, and a lysine amino acid residue, respectively, orderivatives thereof, and L is preferably a pteroyl or folate and Rx eachcomprises an independently selected solubilizing group that isoptionally absent.

Wherein the Tyr, Cys, Ser, and Lys groups indicate a tyrosine, acysteine, a serine, and a lysine amino acid residue, respectively, orderivatives thereof, and L is preferably a pteroyl or folate.Preferably, L is pteroyl.

In specific preferred embodiments the disclosure provides a compound ofFormula I(a), wherein Tyr is selected from the group consisting of:

Suitably, the compounds disclosed herein have a maximum light absorptionwavelengths in the near infrared region of between about 650 nm and 1000nm, for example and preferably, at approximately 800 nm.

In specific preferred embodiments, the compounds disclosed hereininclude a ligand (L) that is effective to target the compound to aparticular cell or tissue type and allow for imaging of that targetedcell or tissue. It is preferable the L is either pteroyl moiety orfolate moiety and more preferable that L is pteroyl moiety. However, itis contemplated that the skilled person may use some other ligand L totarget the compounds to a particular cell surface protein or receptorprotein of interest. In specific and preferred embodiments, the ligandcomprises pteroyl:

Methods of Use

As noted herein above, there is a need for near infrared dye compoundsthat specifically target to regions within a tissue. This is so that thecompounds may be used in imaging techniques and to assist in thediagnosis and therapeutic intervention of disease. As discussed indetail above, the compounds provided herein are useful as dyes andimaging agents in the NIR region of the light spectrum. As such, thecompounds have broad applicability to any number of imaging, diagnostic,and targeted therapeutic methods.

In specific embodiments, the present disclosure relates to methods thatincorporate at least one of the compounds disclosed herein (e.g., ofFormula I, I(a), I(b), I(c), and/or I(d)).can be used to specificallyand sensitively identify tumors within a tissue. More specifically, theidentified tumors may then be therapeutically resected through surgicalmethods. In this manner, the compounds of the present disclosure may beuseful in fluorescence guided surgical resection of tumors, lymph nodes,and the like. Alternatively, the compounds of the present disclosure mayreadily be used in whole body imaging in which the compound isadministered to a subject and the localization of the fluorescencefacilitates identification of a tumor site.

In this manner, the compounds of the present disclosure can be used forthe in vivo identification of diseased tissue in a subject in needthereof. The disclosure method includes irradiating an in vivo body partof the subject containing diseased tissue with light having at least oneexcitation wavelength in the near infrared range from about 600 nm toabout 1000 nm. Fluorescence emanating from a compound of the presentdisclosure administered to the subject and which has specifically boundto and/or been taken up by the diseased tissue in the body part, inresponse to the at least one excitation wavelength is directly viewed todetermine the location and/or surface area of the diseased tissue in thesubject.

Light having a wavelength range from 600 nm and 850 nm lies within thenear infrared range of the spectrum, in contrast to visible light, whichlies within the range from about 401 nm to 500 nm. Therefore, theexcitation light used in practice of the disclosure diagnostic methodswill contain at least one wavelength of light to illuminates the tissueat the infrared wavelength to excite the compounds in order that thefluorescence obtained from the area having uptake of the compounds ofthe present disclosure is clearly visible and distinct from theauto-fluorescence of the surrounding tissue. The excitation light may bemonochromatic or polychromatic. In this manner, the compounds of thepresent disclosure are advantageous as they eliminate the need for useof filtering mechanisms that would be used to obtain a desireddiagnostic image if the fluorescent probe is one that fluoresces atwavelengths below 600 nm. In this manner, the compounds of the presentdisclosure avoid obscured diagnostic images that are produced as aresult of excitation light of wavelengths that would be reflected fromhealthy tissue and cause loss of resolution of the fluorescent image.

Operating rooms for surgical procedures can be equipped with an overheadlight that produces wavelengths of light in the optical emittingspectrum useful in practice of disclosure diagnostic methods, such as alamps that produce light in the appropriate wavelength. Such a light canbe utilized in the practice of the disclosure diagnostic methods merelyby turning out the other lights in the operating room (to eliminateextraneous light that would be visibly reflected from tissue in the bodypart under investigation) and shining the excitation light of nearinfrared wavelength into the body cavity or surgically created openingso that the fluorescent image received directly by the eye of theobserver (e.g., the surgeon) is predominantly the fluorescent imageemanating from the fluorophore(s) in the field of vision. Lightemanating from a source in the 600 nm and 850 nm range, preferably 750nm-850 nm range would be used in accomplishing the goal of directvisualization by the observer so that light reflecting from the bodypart, other than that from the fluorescing moiet(ies), is minimized oreliminated.

Accordingly, in disclosure diagnostic methods, the diseased tissue (andbound or taken-up targeting construct) is “exposed” to the excitationlight (e.g, by surgically created opening or endoscopic delivery of thelight to an interior location. The disclosed method is particularlysuited to in vivo detection of diseased tissue located at an interiorsite in the subject, such as within a natural body cavity or asurgically created opening, where the diseased tissue is “in plain view”(i.e., exposed to the human eye) to facilitate a procedure of biopsy orsurgical excision of the area that has been highlighted by uptake of thecompounds of the present disclosure. As the precise location and/orsurface area of the tumor tissue are readily determined by the uptake ofthe compounds of the present disclosure, the methods employing thecompounds of the present disclosure provide a valuable guide to thesurgeon, who needs to “see” in real time the exact outlines, size, etc.of the mass to be resurrected as the surgery proceeds.

Thus, in specific embodiments, the present disclosure entails opticalimaging of a biological tissue that expresses a folate receptor bycontacting the tissue with a composition comprising compounds of thepresent disclosure (e.g., compounds of Formula I) and allowing time forthe compound in the composition to distribute within the tissue andinteract with the site of folate receptor. After a sufficient time forsuch interaction has passed, the tissue is illuminated with anexcitation light to cause the compound in the composition to fluoresce.The fluorescence is then detected as and where such fluorescence isobserved is an area that contains the folate receptor.

In like manner, the compounds of the present disclosure are used toidentify a target cell type in a biological sample by contacting thebiological sample with such compounds for a time and under conditionsthat allow for binding of the compound to at least one cell of thetarget cell type. The bound compound is then optically detected suchthat presence of fluorescence of the near infrared wavelength emanatingfrom the bound, targeted compound of the present disclosure indicatedthat the target cell type is present in the biological sample. Thismethod thus provides an image of the targeted cell type in the tissuebeing assessed. Most preferably, the targeted cell type is a tumor cellor a lymph node to which a tumor cell has spread.

These methods advantageously provide an improved method of performingimage guided surgery on a subject as the administration of a compositioncomprising the compound of the disclosure under conditions and for atime sufficient for said compound to accumulate at a given surgical sitewill assist a surgeon in visualizing the tissue to be removed.Preferably the tissue is a tumor tissue and illuminating the compoundthat has been taken up by the tissue facilitates visualization of thetumor by the near infrared fluorescence of the compound using infraredlight. With the aid of the visualization facilitated by the targeting ofthe compound of the disclosure to the tumors site, surgical resection ofthe areas that fluoresce upon excitation by infrared light allows animproved and accurate removal of even small tumors.

It should be understood that in any of the surgical methods of thedisclosure the compounds of the present disclosure may be administeredbefore the surgical incision takes place or even after the surgicalcavity and site of the tumor has been revealed by the surgery.

If the putative diseased site is a natural body cavity or surgicallyproduced interior site, an endoscopic device can be optionally used todeliver the excitation light to the site, to receive fluorescenceemanating from the site within a body cavity, and to aid in formation ofa direct image of the fluorescence from the diseased tissue. Forexample, a lens in the endoscopic device can be used to focus thedetected fluorescence as an aid in formation of the image. As usedherein, such endoscope-delivered fluorescence is said to be “directlyviewed” by the practitioner and the tissue to which the targetingconstruct binds or in which it is taken up must be “in plain view” tothe endoscope since the light used in the disclosure diagnosticprocedure will not contain wavelengths of light that penetrate tissue,such as wavelengths in the near infrared range. Alternatively, theexcitation light may be directed by any convenient means into a bodycavity or surgical opening containing a targeting construct administeredas described herein and the fluorescent image so produced can bedirectly visualized by the eye of the observer without aid from anendoscope. With or without aid from any type of endoscopic device, thefluorescent image produced by the disclosure method is such that it canbe viewed without aid of an image processing device, such as a CCDcamera, TV monitor, photon collecting device, and the like.

It is contemplated that the diagnostic or imaging methods of the presentdisclosure allow the surgeon/practitioner to contemporaneouslysee/view/visualize diseased or abnormal tissue through a surgicalopening to facilitate a procedure of biopsy or surgical excision. As thelocation and/or surface area of the diseased tissue are readilydetermined by the diagnostic procedure of the disclosure employing thecompounds described herein, the disclosure method is a valuable guide tothe surgeon, who needs to know the exact outlines, size, etc. of themass, for example, for resection as the surgery proceeds. In particular,it is noted that the compounds of the disclosure fluorescence in thenear infrared range to a greater intensity than those previouslydescribed. As such, advantageously, it is contemplated that less of thecompound will be needed to achieve diagnostic imaging. In addition, thecompounds of the present disclosure penetrate deep into the tumor andhence the disclosure advantageously allows a greater accuracy that thetumor has been removed.

The present disclosure provides methods for utilizing a diagnosticprocedure during surgery in a subject in need thereof by administeringto the subject a composition comprising a compound of the presentdisclosure and irradiating an in vivo body part of the subjectcontaining diseased tissue with light having at least one excitationwavelength in the range from about 600 nm to about 850 nm, directlyviewing fluorescence emanating from a targeting construct administeredto the subject that has specifically bound to and/or been taken up bythe diseased tissue in the body part, wherein the targeting constructfluoresces in response to the at least one excitation wavelength,determining the location and/or surface area of the diseased tissue inthe subject, and removing at least a portion of the tumor tissue.

In yet another embodiment, the present disclosure provides methods forin vivo diagnosis of tumor tissue in a subject in need thereof. In thisembodiment, the disclosure method comprises contacting samples of tumorcells obtained from the subject in vitro with a plurality of detectablylabeled compounds, each of which binds to or is selectively taken up bya distinct tumor type, determining which of the compounds is bound to ortaken up by the sample tumor cells, administering a diagnosticallyeffective amount of at least one biologically compatible fluorescingtargeting construct containing a compound of the present disclosure thathas been determined to bind to and/or be taken up by the sample tumorcells and a fluorophore responsive to at least one wavelength of lightin the range from about 600 nm to about 850 nm, and diagnosing thelocation and/or surface area of the tumor tissue in the in vivo bodypart by directly viewing fluorescence emanating from the targetingconstruct bound or taken up in the tumor tissue upon irradiation thereofwith light providing the at least one excitation wavelength for thefluorescent targeting construct.

In some embodiments, a single type of fluorescent moiety is relied uponfor generating fluorescence emanating from the irradiated body part(i.e., from the fluorescent targeting construct that binds to or istaken up by diseased tissue) and subjecting the targeting construct witha source of light from the near infrared spectrum.

In other embodiments, it is contemplated that a plurality. (i.e., two,three, four, or more) targeting constructs are used to obtain adiagnostic image. Such additional targeting constructs may be additionalcompounds of the present disclosure distinct from the first suchcompound. Alternatively, the additional targeting constructs maycomprise the dyes described herein but with the pteroyl moiety beingreplaced by a ligand for another receptor other than folate receptor. Instill other embodiments, the additional targeting moieties may be otherfluorescing targeting constructs (e.g., antibodies, or biologicallyactive fragments thereof, having attached fluorophores) that bind toother receptors or antigens on the tumor or tissue (e.g., a site ofatherosclerosis, infection, cardiovascular diseases, neurodegenerativediseases, immunologic diseases, autoimmune diseases, respiratorydiseases, metabolic diseases, inherited diseases, infectious diseases,bone diseases, and environmental diseases or the like) to be imaged. Anyadditional targeting moiety that specifically targets the tumor orspecific site on the tissue may be used provided that it is specific forthe site to be monitored. The purpose of the additional fluorescingtargeting construct is to increase the intensity of fluorescence at thesite to be monitored thereby aiding in detection of diseased or abnormaltissue in the body part. For example, a given tumor may have numerousmarkers and in addition to the compounds of the present disclosure acocktail of fluorescent moieties is provided which are specific for thatgiven tumor such that the signal emanating from the tumor is generatedby more than one compound or fluorescent moiety that has targeted andbecome localized to the tumor site of interest.

In practice, the skilled person would administer a compound of thepresent disclosure either alone or as part of a cocktail of targetingdetectable moieties and allow these compounds and targeting moieties tobind to and/or be taken up by any targeting tissue that may be presentat the site under investigation and then provide a supply of the lightsource. Typically, the compounds of the present disclosure and anyadditional targeting moieties will be administered prior to surgery fora time and in compositions that allow the fluorescent compounds of thepresent disclosure as well as any additional fluorescent constructs tobe taken up by the target tissue.

Those of skill in the art will be able to devise combinations ofsuccessively administered fluorescing targeting constructs, each ofwhich specifically binds to the target site. It is preferable that allof the fluorescing targeting constructs used in such cocktails toidentify the target tissue comprise fluorophores that fluoresce withinthe same wavelength band or at the same wave length as does the compoundof the present disclosure (e.g. a fluorescing sensitive to near infraredwavelength of light in the compounds of the present disclosure) tominimize the number of different light sources that need to be employedto excite simultaneous fluorescence from all of the different targetingconstructs used in practice of the disclosure method. However, it iscontemplated that the additional targeting moieties other than thecompounds of the present disclosure may fluorescence in response to theirradiating light at a different color (i.e., has a differentwavelength) than that from the florescent compounds of the presentdisclosure. The difference in the colors of the fluorescence emanatingfrom the compounds of the present disclosure and those of the additionaltargeting compounds may aid the observer in determining the location andsize of the diseased tissue. In some examples, it may be desirable toinclude fluorophores in targeting constructs targeted to target normaltissue and the compounds of the present disclosure to target diseasedtissue such that the contrast between the diseased tissue and normaltissue is further enhanced to further aid the observer in determiningthe location and size of the target tissue. The use of such additionalfluorophores and targeting agents in addition to the compounds of thepresent disclosure provides the advantage that any natural fluorescenceemanating from normal tissue is obscured by the fluorescence emanatingfrom fluorophore(s) in supplemental targeting constructs targeted to thenormal tissue in the body part. The greater the difference in colorbetween the fluorescence emanating from normal and target tissue, theeasier it is for the observer to visualize the outlines and size of thetarget tissue. For instance, targeting a fluorescing targeting constructcomprising a fluorophore producing infrared light from the compounds ofthe present disclosure to the target tissue (i.e., abnormal tissue) anda fluorophore producing green light to healthy tissue aids the observerin distinguishing the target tissue from the normal tissue. Those ofskill in the art can readily select a combination of fluorophores thatpresent a distinct visual color contrast.

The spectrum of light used in the practice of the disclosure method isselected to contain at least one wavelength that corresponds to thepredominate excitation wavelength of the targeting construct, or of abiologically compatible fluorescing moiety contained within thetargeting construct. Generally the excitation light used in practice ofthe disclosure method comprises at least one excitation wavelength oflight in the near infrared wavelength range from about 600 nm to about850 nm

However, when a combination of targeting ligands that fluoresce atdifferent wavelengths is used in practice of the disclosure, thespectrum of the excitation light must be broad enough to provide atleast one excitation wavelength for each of the fluorophores used. Forexample, it is particularly beneficial when fluorophores of differentcolors are selected to distinguish normal from diseased tissue, that theexcitation spectrum of the light(s) includes excitation wavelengths forthe fluorophores targeted to normal and target tissue.

As noted herein the compounds of the present disclosure are specificallytargeted to the folate receptor by way of pteroyl or folate ligand beingpart of the compounds of the present disclosure. In embodiments where anadditional targeting moiety is used, the targeting construct of such anadditional targeting moiety is selected to bind to and/or be taken upspecifically by the target tissue of interest, for example to an antigenor other surface feature contained on or within a cell thatcharacterizes a disease or abnormal state in the target tissue. As inother diagnostic assays, it is desirable for the targeting construct tobind to or be taken up by the target tissue selectively or to an antigenassociated with the disease or abnormal state; however, targetingconstructs containing ligand moieties that also bind to or are taken upby healthy tissue or cell structures can be used in the practice of thedisclosure method so long as the concentration of the antigen in thetarget tissue or the affinity of the targeting construct for the targettissue is sufficiently greater than for healthy tissue in the field ofvision so that a fluorescent image representing the target tissue can beclearly visualized as distinct from any fluorescence coming from healthytissue or structures in the field of vision.

For example, colon cancer is often characterized by the presence ofcarcinoembryonic antigen (CEA), yet this antigen is also associated withcertain tissues in healthy individuals. However, the concentration ofCEA in cancerous colon tissue is often greater than is found in healthytissue, so an anti-CEA antibody could be used as a ligand moiety in thepractice of the disclosure. In another example, deoxyglucose is taken upand utilized by healthy tissue to varying degrees, yet its metabolism inhealthy tissues, except for certain known organs, such as the heart, issubstantially lower than in tumor. The known pattern of deoxyglucoseconsumption in the body can therefore be used to aid in determination ofthose areas wherein unexpectedly high uptake of deoxyglucose signals thepresence of tumor cells.

The disease or abnormal state detected by the disclosure method can beany type characterized by the presence of a known target tissue forwhich a specific binding ligand is known. For example, various heartconditions are characterized by production of necrotic or ischemictissue or production of artherosclerotic tissue for which specificbinding ligands are known. As another illustrative example, breastcancer is characterized by the production of cancerous tissue identifiedby monoclonal antibodies to CA15-3, CA19-9, CEA, or HER2/neu. It iscontemplated that the target tissue may be characterized by cells thatproduce either a surface antigen for which a binding ligand is known, oran intracellular marker (i.e. antigen), since many targeting constructspenetrate the cell membrane. Representative disease states that can beidentified using the disclosure method include such various conditionsas different types of tumors, bacterial, fungal and viral infections,and the like. As used herein “abnormal” tissue includes precancerousconditions, necrotic or ischemic tissue, and tissue associated withconnective tissue diseases, and auto-immune disorders, and the like.Further, examples of the types of target tissue suitable for diagnosisor examination using the disclosure method include cardiac, breast,ovarian, uterine, lung, endothelial, vascular, gastrointestinal,colorectal, prostatic tissue, endocrine tissue, and the like, as well ascombinations of any two or more thereof.

Simply by way of example, antigens for some common malignancies and thebody locations in which they are commonly found are known to those ofskill in the art, and targeting ligands, such as antibodies or for theseantigens or indeed ligands where the antigens are receptors are known inthe art. For example, CEA (carcinoembryoinc antigen) is commonly foundin tumors from the colon, breast and lung; PSA (prostate specificantigen, or sometimes referred to as prostate specific membrane antigen(PSMA)) is specific for prostate cancer; CA-125 is commonly found intumors of ovarian cancer origin, CA 15-3, CA19-9, MUC-1, Estrogenreceptor, progesterone receptor and HER2/neu are commonly found inbreast cancer tumors, alpha-feto protein is found in both testicularcancer and hepatic cancer tumors, beta-human chorionic gonadotropin isfound testicular cancer and choriocarcinoma, both estrogen receptor andprogesterone receptor also are found in uterine cancer tumors andepidermal growth factor receptor is commonly found in tumors frombladder cancer. Other tumor specific ligands and markers are well knownto those of skill in the art. In preferred embodiments, the presentdisclosure employs folate or pteroyl moieties for targeting the folatereceptor and PMSA target moieties for targeting the dyes to prostatecancer cells.

It is contemplated that any of these commonly known markers of tumorscan be targeted either using the dyes described herein (by switching outthe pteroyl moiety for a moiety that specifically targets these markers)or alternatively, these markers can be targeted in addition and incombination with the folate receptor that is being targeted using thecompounds of the present disclosure. As discussed previously, it may beparticularly advantageous to have targeting moieties to severaldifferent markers on a given tumor to serve as a diagnostic cocktail inwhich several markers are targeted to more brightly and clearlyvisualize the tumor.

In addition to chemical compounds, the targeting moieties in suchcocktails may include a protein or polypeptide, such as an antibody, orbiologically active fragment thereof, preferably a monoclonal antibody.The supplemental fluorescing targeting construct(s) used in practice ofthe disclosure method may also be or comprise polyclonal or monoclonalantibodies tagged with a fluorophore. The term “antibody” as used inthis disclosure includes intact molecules as well as functionalfragments thereof, such as Fab, F(ab′)2, and Fv that are capable ofbinding the epitopic determinant. Methods of making these fragments areknown in the art. (See for example, Harlow & Lane, Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory, New York, 1988,incorporated herein by reference). As used in this disclosure, the term“epitope” means any antigenic determinant on an antigen to which theparatope of an antibody binds. Epitopic determinants usually consist ofchemically active surface groupings of molecules such as amino acids orsugar side chains and usually have specific three dimensional structuralcharacteristics, as well as specific charge characteristics.

In addition to antibodies, the cocktails may comprise compounds in whichthe ligand moiety attached to the fluorescent targeting construct isselected from among the many biologically compatible compounds that bindwith specificity to receptors and/or are preferentially taken up bytumor cells, and can be used as the ligand moiety in the disclosuretargeting constructs. Compounds that are preferentially “taken up” bytumor cells may enter the cells through surface or nuclear receptors(e.g., hormone receptors), pores, hydrophilic “windows” in the celllipid bilayer, and the like.

Illustrative of this class of compounds to target tumors aresomatostatin, somatostatin receptor-binding peptides, deoxyglucose,methionine, and the like. Particularly useful somatostatinreceptor-binding peptides are a long-acting, octapeptide analog ofsomatostatin, known as octreotide(D-phenylalanyl-L-cysteinyl-L-phenylala-nyl-D-tryptophyl-L-lysyl-L-threonyl-N-[2-hydroxy-1-(hydroxymethyl)propyl]-L-cysteinamidecyclic (2→7)-disulfide), lanreotide, an oral formulation of octreotide,P829, P587, and the like. Somatostatin-binding peptides are disclosed inU.S. Pat. No. 5,871,711, and methods for linking such peptidescovalently to a radioisotope through their carboxyl terminal amino acidunder reducing conditions are disclosed in U.S. Pat. No. 5,843,401,which are both incorporated herein by reference in their entireties. Oneof skill in the art can readily adapt such teachings for the preparationof fluorescence-sensitive somatostatin receptor-binding peptides bysubstituting the fluorescing moieties of this disclosure in the place ofa radioisotope.

Somatostatin and somatostatin receptor-binding peptides are particularlyeffective for use as the tumor-targeting ligand moiety in the targetingconstruct when the disease state is a neuroendocrine or endocrine tumor.Examples of neuroendocrine tumors that can be diagnosed using thedisclosure method include adenomas (GH-producing and TSH-producing),islet cell tumors, carcinoids, undifferentiated neuroendocrinecarcinomas, small cell and non-small cell lung cancer, neuroendocrineand/or intermediate cell carcinomas, neuroendocrine tumors of ovary,cervix, endometrium, breast, kidney, larynx, paranasal sinuses, andsalivary glands, meningiomas, well differentiated glia-derived tumors,pheochromocytomas, neuroblastomas, ganglioneuro(blasto)mas,paragangliomas, papillary, follicular and medullary carcinomas inthyroid cells, Merkel cell carcinomas, and melanomas, as well asgranulomas and lymphomas. These tumor cells are known to havesomatostatin receptors and can be targeted using somatostatin orsomatostatin receptor binding peptides as the tumor-targeting ligand inthe disclosure fluorescent targeting construct.

Vasointestinal peptide (VIP), which is used in VIP receptor scintigraphy(I. Virgolini, Eur J. Clin. Invest. 27(10):793-800, 1997, is also usefulin the disclosure method for diagnosis of small primary adenocarcinomas,liver metastases and certain endocrine tumors of the gastrointestinaltract.

Another molecule illustrative of the tumor-targeting ligands that arepreferentially taken up by tumors is deoxyglucose, which is known to bepreferentially taken up in a variety of different types of tumors.Illustrative of the types of tumors that can be detected usingdeoxyglucose as the tumor-targeting ligand include melanoma, colorectaland pancreatic tumors, lymphoma (both HD and NHL), head and neck tumors,myeloma, cancers of ovary, cancer, breast, and brain (high grade andpituitary adenomas), sarcomas (grade dependent), hepatoma, testicularcancer, thyroid (grade dependent) small cell lung cancer, bladder anduterine cancer, and the like.

Yet other tumor-targeting compounds that can be used in cocktails of thepresent disclosure include 1-amino-cyclobutane-1-carboxylic acid andL-methionine. L-methionine is an essential amino acid that is necessaryfor protein synthesis. It is known that malignant cells have alteredmethionine metabolism and require an external source of methionine.

Additional examples of biologically compatible tumor-targeting compoundsthat bind with specificity to tumor receptors and/or are preferentiallytaken up by tumor cells include mammalian hormones, particularly sexhormones, neurotransmitters, and compounds expressed by tumor cells tocommunicate with each other that are preferentially taken up by tumorcells, such as novel secreted protein constructs arising fromchromosomal aberrations, such as transfers or inversions within theclone.

Hormones, including sex hormones, cell growth hormones, cytokines,endocrine hormones, erythropoietin, and the like also serve well astumor targeting moieties. As is known in the art, a number of tumortypes express receptors for hormones, for example, estrogen,progesterone, androgens, such as testosterone, and the like. Suchhormones are preferentially taken up by tumor cells, for example, viaspecific receptors.

The targeting constructs and supplemental targeting constructs used inpractice of the disclosure method can be administered by any route knownto those of skill in the art, such as topically, intraarticularly,intracisternally, intraocularly, intraventricularly, intrathecally,intravenously, intramuscularly, intraperitoneally, Experimental Section:

Materials: N¹⁰-Trifluroacetylpteroic acid [Pte-N¹⁰-(TFA)-OH] (IrvineChemistry Lab, Anaheim, Calif.). O-t-Butyl-L-tyrosine t-butyl esterhydrochloride and0-(7-azabenzo-triazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU) (Chem-Impex Int., Chicago, Ill.). All otherchemicals, cell culture materials, and animal supplies were obtainedfrom major suppliers.

General methods: Moisture and oxygen sensitive reactions were carriedout under an argon atmosphere. Solid phase peptide synthesis (SPPS) wasperformed using a standard peptide synthesis apparatus (Chemglass,Vineland, N.J.). Flash chromatography was conducted with silica gel asthe solid phase, and TLC was performed on silica gel TLC plates (60f254, 5×10 cm) and visualized under UV light. All peptides and peptideconjugates were purified by preparative reverse phase (RP)-HPLC (Waters,xTerra C18 10 μm; 19×250 mm) and analyzed UPLC (Acquity, BEH C18 1.7 μm;2.1×50 mm) using solvent A=10 mM Ammonium acetate (NH₄OAc; pH=7.0) andsolvent B=Acetonitrile (CH₃CN). ¹H and ¹³C spectra were acquired with aBruker 500 MHz and 125 MHz NMR spectrometer equipped with a TXIcryoprobe. Samples were run in 5 mm NMR tubes using DMSO-d6, orDMSO-d₆/D₂O. Pre-saturation was used to reduce the intensity of theresidual H₂O peak. All ¹H signals are recorded in ppm with reference toresidual trichloromethane (also known as chloroform or CHCI3) (7.27 ppm)or dimethyl sulfoxide (DMSO) (2.50 ppm), and data are reported ass=singlet, d=doublet, t=triplet, q=quartet, and m=multiplet orunresolved, b=broad, with coupling constants in Hz.

LC/MS analyses were obtained using a Waters micromass ZQ 4000 massspectrometer coupled with a UV diode array detector. High resolutionmass spectrometric results were obtained by matrix-assisted laserdesorption ionization (MALDI) mass using an Applied Biosystems(Framingham, Mass.) Voyager DE PRO mass spectrometer. This instrumentutilizes a nitrogen laser (337 nm UV laser) for ionization with atime-of-flight mass analyzer. The matrix used for these samples wasalpha-cyano-4-hydroxy cinnamic acid and the peptide LHRH was used asinternal standard.

KB cells [a derivative of HeLa cells (a human cervical cancer cellline)], IGROV cells (a human ovarian cancer cell line), SKOV3 cells (ahuman ovarian cancer cell line), OVCAR3 cells (a human ovarian cancercell line), HCC827 (a human non-small cell lung cancer cell line),MDA-MB231 (a human breast cancer cell line), M109 murine lung cancercell line), and A549 cells (a alveolar basal epithelial carcinoma cellline) were obtained from American Type Culture Collection (ATCC)(Rockville, Md.) and grown as a monolayer and L1210 (a human leukemiacell line) grown as suspension using folate free or normal 1640RPMI-1640 medium (Gibco, N.Y.) containing 10% heat-inactivated fetalbovine serum (Atlanta Biological, GA) and 1% penicillin streptomycin(Gibco, N.Y.) in a 5% carbon dioxide: 95% air-humidified atmosphere at37° C. for at least six passages before they were used for the assays.

Athymic female nude (nu/nu) and Balb/C mice (5 weeks old, 18-20 g) werepurchased from Harlan (Indianapolis, Ind.) and maintained ongamma-irradiated folate-deficient special diet (Teklad, Wis.) for atleast 2 weeks before start of the study. Animals were housed with fivemice per cage (5/cage) in a barrier, pathogen-free cloaked rack.Autoclaved tap water and food were given as needed. The animals werehoused in a sterile environment on a standard 12 hour light-dark cyclefor the duration of the study. Mice were identified individually by earpunch.

In Vitro Binding:

Column 88, lines 10-31 of U.S. Pat. No. 9,061,057 are expresslyincorporated by reference.

Flow Cytometry: KB (a human cervical cancer cell line), IGROV (a humanovarian cancer cell line), SKOV3 (a human ovarian cancer cell line),OVCAR3 (a human ovarian cancer cell line), HCC827 (a human lung cancercell line), MDA-MB231 (a human triple negative breast cancer cell line),M109 (a murine lung cancer cell line), A549 (a human lung cancer cellline), L1210 (a murine leukemia cancer cell line) cells were seeded intoa T75 flask and allowed to form a monolayer over 24 hours and L1210cells were grown in suspension. After trypsin digestion, release cellswere transferred into centrifuge tubes (1×10⁶ cells/tube) andcentrifuged. The medium was replaced with fresh medium containing thecompound of interest (100 nM) in the presence or absence of 100-foldexcess FA and incubated for 1 hour at 37° C. After rinsing twice with 1mL of fresh medium (2×1.0 mL) and once with 1 mL of PBS (1×1.0 mL),cells were resuspended in 1 mL of PBS and cell bound fluorescence wasanalyzed (100,000 cells/sample) using a flow cytometer (Cytomics F500,Beckman Coulter). Untreated cells in PBS served as a negative control.

Confocal Microscopy: KB, IGROV, SKOV3, MDA-MB231, or A549 cells (50,000cells/well in 1 mL) were seeded into poly-D-lysine microwell Petridishes and allowed cells to form monolayers over 12 hours. Spent mediumwas replaced with fresh medium containing 100 nM of the compound ofinterest (100 nM) in the presence or absence of 100-fold excess FA andcells were incubated for 1 hour at 37° C. After rinsing twice with 1.0mL fresh medium (2×1.0 mL) and once with 1.0 mL PBS (1×1.0 mL), confocalimages were acquired using a confocal microscopy (FV 1000, Olympus).

Whole Body Imaging: (a) Xenograft Model:

Seven-week-old female nu/nu mice were inoculated subcutaneously with KB(1.0×10⁶/mouse in RPMI1640 medium), IGROV (5.0×10⁶/mouse in 50% highconcentrated matrigel+RPMI1640 medium), SKOV3 (5.0×10⁶/mouse in RPMI1640medium), OVCAR3 (5.0×10⁶/mouse in RPMI1640 medium), MDA-MB231(1.0×10⁶/mouse in RPMI1640 medium), M109 (1.0×10⁶/mouse in RPMI1640medium) or A549 cells (2.0×10⁶/mouse in RPMI1640 medium) on theshoulder. Growth of the tumors was measured in perpendicular directionsevery 2 days using a caliper (body weights were monitored on the sameschedule), and the volumes of the tumors were calculated as 0.5×L×W²(L=longest axis and W=axis perpendicular to L in millimeters). Oncetumors reached approximately 300-400 mm³ in volume, animals (3-5mice/group) were intravenously injected with the compound of interest inPBS (for dose escalation in KB tumor model: 0.3-90 nmol, for timedependent in KB tumor model: 2 or 10 nmol, and for general whole bodyimaging and biodistribution for all tumor models: 2 nmol, in 100 μL inPBS per mouse). For whole body imaging and biodistribution studies,animals were euthanized 2 hours after administration of the compound ofinterest by CO₂ asphyxiation. For time dependent studies, animals wereimaged under anesthesia using isoflurane. Whole body imaging (intacttumor) experiments was then performed using a Caliper IVIS Lumina IIImaging Station with Living Image 4.0 software (PerkinElmer Inc, MA).

(b) Orthotopic Models:

Ovarian cancer was developed through surgical implantation of ovariancancer cell lines near the ovarian bursa of the mouse. Seven-week-oldfemale athymic nude mice were given 1-5% isoflurane for anesthesia andsubcutaneous injection of 5 mg/kg meloxicam preoperatively foranalgesia. The mice were placed dorsal side up and washed above theright ovary (just lateral to the epaxial muscles and ˜1.15 cm caudal tothe rib cage) with a chlorhexidine scrub to ensure a sterile area forincision. After an insertion was made using scalpel through the skin,the peritoneal lining was lifted to make a small incision using ascissor and widened using a forceps. The ovarian fat pad was carefullylifted out and placed on a wetted gauze pad adjacent to the incision.Ovarian cancer cells (50,000 cells in 5 μL) were injected between thebursa and the ovary using a 30 gage needle. After placing the ovarianfat pad back into the body cavity, the body wall was closed using 3-0 or4-0 vicryl and the skin was closed using staples.

Breast cancer was developed by implanting breast cancer cells in thememory fat pad of the mouse.

(c) Metastatic Models:

One million (1×10⁶) of either L1210A or MDA-MB231 cancer cells per mouse(cancer cells/mouse) were administered via tail vein injection into themice. Tumor growth was monitored every other day using a Caliper IVISLumina II Imaging Station with Living Image 4.0 software (PerkinElmerInc.).

Tissue Distribution:

Following whole body imaging, animals were dissected and selectedtissues (heart, lung, liver, spleen, kidneys, stomach, small intestine,large intestine, muscle, skin, and tumor) were analyzed for fluorescenceactivity using IVIS imager using same settings as in whole body imaging.

Example 1: Design and Synthesis

A synthesis of compound 4 (OTL38) is shown in Scheme 1.N¹⁰-Trifluroacetylpteroic acid was first reacted with tert-butylprotected tyrosine in the presence of HATU, an acid activating group, togenerate compound 2 in quantitative yield. The compound 2 wasprecipitated under mild acidic conditions and then treated with acocktail of trifluroacetic acid, tri-isopropylsilane, and water tode-protect tertiary butyl groups from the acid and hydroxyl group. Afterprecipitating the compound 3 in ether, it was dissolved in water at pH9.5 to produce compound 4.

Example 2. Comparison of Chemical and Optical Properties of Compound 4with Commercially Available NIR Fluorophores

TABLE 1 Comparison of chemical and optical properties of NIR dyesProperty CW800 ZW80o LS288 Kodak ICG OTL38 CY7 Cy7.5 Alx750 DyL750 MW(Da) 1091 1149 973 1117 775 1326 549.8 649.9 ~1300 1092 E (M⁻¹ cm⁻¹)237000 249000 122000 224000 121000 272000 240600 223000 290000 220000λem (nm) 786 772 761 802 807 776 750 788 753 754 λex (nm) 800 788 778818 822 796 773 808 782 776 Stokes Shift (nm) 14 16 17 16 15 20 23 20 2922 Q (%) 14.2 15.1 11.2 12.5 9.3 15.6 Cost/g ($) 30,000 50,000 1,500<500 12,000 12,000 50,000 50,000

Example 3. In Vitro Binding Affinity and Specificity in Human CervicalTumor Xenograft Model

Overexpression of FR-α in KB cells (a derivative of HeLa cell line andHeLa cells are human cervical cancer cell line) is well established inthe literature. Therefore, all the initial optimizations were done usingKB cells. Optimized conditions were used in the ovarian cancerxenografts and orthotopic modes.

Example 6 (Column 71, line 44 to Column 72, line 35) of U.S. Pat. No.9,061,057 is expressly incorporated by reference with compound 6described in the '057 patent as OTL-0038.

Example 4. Binding and Internalization of Compound 4 to Human CervicalCancer and Triple Negative Breast Cancer Cells by Confocal Microscopy

The binding and internalization of OTL38 is illustrated in FIGS. 3A, 4B,4D, and 4E. KB, MDA-MB231 and SKOV3 cells each independently expressFR-alpha while A549 cells do not.

Example 5. In Vivo Efficacy of OTL38 in a Human Cervical CancerXenograft Model that Express High Levels of FR-α

Analysis of whole body imaging demonstrated that OTL38 accumulatedpredominantly in the FR positive KB tumors, with no substantialfluorescence activity in the other tissues (FIG. 4A). Analysis of tissuebiodistribution was performed on the same animals that were subjected towhole body imaging by euthanizing each mouse, removing their organs andimaging using IVIS imager. The highest fluorescence intensity wasobserved in FR-positive tumors with no accumulation in the other tissuesexcept the kidneys and skin (FIG. 4B). All FR-negative tissues displayedvery low uptake levels of OTL38 resulting in excellent tumor-to-tissueratios except the skin (FIG. 4C). Uptake of OTL38 in the kidneys wasanticipated, since the apical membrane of the proximal tubule of thekidney has been known to express high levels of FR. Moreover, it'spossible that the compound 4 is excreted through the kidneys due totheir low molecular weights and half-life (most of the folate conjugateshave <30 min half-life). Fluorescence in the skin can be due toreflection of the light from the skin and/or skin tissue of the nudemice may have higher level of immune cells such as monocytes,macrophages, etc. These immune cells express FR beta and OTL38 binds toFR-alpha and beta with same affinity. On the other hand, the dyecomponent of OTL38 may non-specifically bind to the nude mice skintissue with slow clearance at 2 h time point.

Following 24 hours post injection of OTL38, tumor-to-skin ratio of a KBtumor bearing mouse was further evaluated using ImageJ analysis (freesoftware that can be downloaded from NIH website) as illustrated inFIGS. 5A-5E. The whole body image of the mouse was acquired asfluorescence in a yellow-red scale (FIG. 5A) and a gray scale (FIGS. 5Band D). A line (FIG. 5B) or a box (FIG. 5D) was drawn across the skinand tumor, and the tumor-to-skin ratio was analyzed using a plot of thefluorescence gray value vs. distance. As shown in the FIGS. 5C and E,fluorescence intensity in the skin is minimal at the 24 h time pointcompared to fluorescence intensity in the tumor, indicating that OTL38has minor nonspecific skin tissue uptake (most probably either OTL38binds to FR beta on the immune cells in the skin and/or there isnon-specific binding of the dye component of the OTL38) that clearswithin 24 h. More importantly, we assume that the fluorescence in theskin, even if there is minor uptake, this should not cause anyinterference during an image guided open surgery for ovarian cancer.

Example 6. Optimal Dose Determination in a Human Cervical CancerXenograft Model that Express High Levels of FR-α

In order to determine the optimal dose of OTL38 that has higher tumoruptake and higher tumor-to-background ratio, a dose dependent study wasconducted by injecting increasing concentrations of OTL38 (0.3, 1.3, 10,20, 30, 60, and 90 nmol) to mice bearing KB tumor xenografts. Animalswere sacrificed, the tissues were harvested, and they were imaged usingthe IVIS imager. As shown in the Table 1 and FIGS. 6(A-H), all the doseshad higher tumor uptake in the folate receptor-positive tumors except0.3 nmol dose. On the other hand, higher kidney uptake was also observedfor dose range 0.3-10 nmol and less kidney uptake (relative to tumoruptake) was observed for dose range 30-90 nmol. Moreover, highernon-specific uptake in healthy tissues was observed at 60 and 90 nmoldoses. OTL38 showed lower uptake in FR-positive tumors (weakfluorescence intensity) at 0.3 nmol dose may be due to incompletesaturation of FR on the tumor cells.

Dose range between 1.0-30.0 nmol showed very good tumor uptake andexcellent tumor-to-normal tissue ratio (FIG. 6I. Higher than 30 nmoldose kidney uptake may be due to the clearance of the probe through thekidneys and expression of folate receptor on kidneys.

Dose levels 60.0 nmol and 90.0 nmol show higher non-specific uptake.However, these dose levels still have high tumor uptake and less kidneyuptake, including 30 nmol dose. Less kidney uptake may be due to thealternative clearance of the probe through liver and gut. Therefore,OTL38 may be forming aggregates at these higher concentrations.

Therefore, we concluded that about 1.0-about 3.0 nmol are the lowestdoses to administer to obtain good tumor-to-background ratio whilemaintaining the non-invasive aspect for tumor imaging (FIG. 6).Moreover, 30 nmol may be the highest dose that could administer toobtain best tumor-to-background ratio. Therefore, we decided to use 2nmol of OTL38 for the rest of the animal studies.

Example 7. Efficacy of in a Human Cervical Cancer Xenograft Model atOptimize Dose that Express High Levels of FR-α

As shown in FIGS. 7A-7C, OTL38 demonstrated an excellent whole bodyimaging (FIG. 7A), tissue biodistribution (FIG. 7B), and tumor-to-tissue(FIG. 7C) at 2 nmol dose at 2 h post injection.

Example 8. Time to Determine Optimal Image in a Human Cervical CancerXenograft Model

In effort to determine the optimal time to image with higher tumoruptake and higher tumor-to-background ratio, time dependent study wasconducted. By administering 10 nmol of OTL38 via tail vein and liveimaging was conducted using IVIS imager from 15 min to 48 h postinjection.

The time dependent study demonstrated that OTL3 accumulated in the FRpositive tumor and kidneys within 1 hour and saturated FR [1.2-1.5×10⁹(p/sec/cm²/sr)/(μW/cm²)] within 2-4 hours. FIG. 8 also shows that OTL38starts clearing from the skin at 2 hour time point and minimalfluorescence was observed at 24-48 hour time points. However,fluorescence in tumor remains high even at 48 hour time point.

Example 9. In Vivo Efficacy of OTL38 in a Human Ovarian Cancer (IGROV, aHuman Ovarian Cancer Cell Line) Model that Express Moderate Levels ofFR-α (a) Xenograft Tumor Model

As seen in the FIG. 9A, OTL38 accumulated mainly in the FR-positiveIGROV tumor xenografts, with no substantial fluorescence activity in theother tissues. To quantitate in vivo specificity of OTL38 to ovariancancer, ex-vivo tissue biodistribution study was performed on the sameanimals that were subjected to whole body imaging. The highestfluorescence intensities were observed in FR-positive tumors and kidneys(FIG. 9B). All the other normal tissues displayed minimal levels or nouptake, resulting in excellent tumor-to-normal tissue ratios (FIG. 9C)especially given IGROV cells express moderate levels of FR-α.

(b) Ovarian Cancer Orthotopic Model

As seen in FIG. 10A, OTL38 accumulated mainly in the FR-positive IGROVorthotopic ovary tumor and there was no substantial fluorescenceactivity in the healthy ovary or the other tissues except kidneysresulting in excellent tumor-to-normal tissue ratios (FIG. 10B). Note:Kidneys were removed from the biodistribution images.

Example 10. In Vivo Efficacy of OTL38 in a Human Ovarian Cancer (SKOV3,a Human Ovarian Cancer Cell Line) Model that Express Low Levels of FR-α(a) Xenograft Tumor Model

As seen in the FIG. 11A, OTL38 accumulated mainly in the FR-positiveSKOV3 tumor xenografts, with no substantial fluorescence activity in theother tissues. To quantitate in vivo specificity of OTL38 to ovariancancer, ex-vivo tissue biodistribution study was performed on the sameanimals that were subjected to whole body imaging. The highestfluorescence intensities were observed in FR-positive tumors and kidneys(FIG. 11B). All the other normal tissues displayed minimal levels or nouptake, resulting in excellent tumor-to-normal tissue ratios (FIG. 11C)especially given SKOV3 cells express low levels of FR-α.

(b) Ovarian Cancer Orthotopic Model

As seen in the FIG. 12A, OTL38 accumulated mainly in the ovary withFR-positive SKOV3 orthotopic tumor and there was no substantialfluorescence activity in the healthy ovary or the other tissues exceptkidneys resulting in excellent tumor-to-normal tissue ratios (FIG. 12C)especially given SKOV3 cells have lower levels of FR-α.

Example 11. In Vivo Efficacy of OTL38 in a Breast Cancer (MDA-MB 231, aHuman Ovarian Cancer Cell Line) Model that Express High Levels of FR-α(a) Xenograft Tumor Model

As seen in the FIG. 13A, OTL38 accumulated mainly in the FR-positiveMDA-MB 231 tumor xenografts, with no substantial fluorescence activityin the other tissues. To quantitate in vivo specificity of OTL38 tobreast cancer, ex-vivo tissue biodistribution study was performed on thesame animals that were subjected to whole body imaging. The highestfluorescence intensities were observed in FR-positive tumors and kidneys(FIG. 13B). All the other normal tissues displayed minimal levels or nouptake, resulting in excellent tumor-to-normal tissue ratios (FIG. 13C).

(b) Orthotopic and Metastatic Models

As seen in FIG. 14A, OTL38 accumulated mainly in the FR-positive MDA-MB231 orthotopic tumors, with no substantial fluorescence activity in theother tissues. To quantitate in vivo specificity of OTL38 to breastcancer, ex-vivo tissue biodistribution study was performed on the sameanimals that were subjected to whole body imaging. The highestfluorescence intensities were observed in FR-positive tumors and kidneys(FIG. 14B). All the other normal tissues including healthy memory padand breast displayed minimal levels or no uptake, resulting in excellenttumor-to-normal tissue ratios (FIG. 14C).

As seen in the FIG. 14 (d), OTL38 mainly accumulated in the breastcancer cells metastasized to lung.

Example 12. In Vivo Efficacy of OTL38 in a Leukemia Cancer Cells(L1210A, a Murine Leukemia Cancer Cell Line) Metastasized to Liver, Lungand Spleen

As seen in the FIGS. 15A and 15B, OTL38 accumulated mainly in theFR-positive L1210A tumors metastasize to the liver, lung and spleen,with no substantial fluorescence activity in the other tissues. Toquantitate in vivo specificity of OTL38 to breast cancer, ex-vivo tissuebiodistribution study was performed on the same animals that weresubjected to whole body imaging. All the other normal tissues includinghealthy memory fat pad and breast displayed minimal levels or no uptake,resulting in excellent tumor-to-normal tissue ratios (FIG. 15C).

Example 13. In Vivo Efficacy of OTL38 in a Lung Cancer Cells (M109, aMurine Lung Cancer Cell Line) Metastasized to Head & Neck

As seen in the FIG. 16A, OTL38 accumulated mainly in the FR-positiveM109 tumors metastasize to the liver, lung and spleen, with nosubstantial fluorescence activity in the other tissues. To quantitate invivo specificity of OTL38 to breast cancer, ex-vivo tissuebiodistribution study was performed on the same animals that weresubjected to whole body imaging. All the other normal tissues includinghealthy memory fat pad and breast displayed minimal levels or no uptake,resulting in excellent tumor-to-normal tissue ratios.

Example 14. In Vivo Specificity of OTL38 for FR

(a) Blocking the FR with Excess Folic Acid

Both whole body imaging (FIGS. 17A and 17B) and tissue biodistributionimages (FIGS. 7C and 7D) demonstrated that OTL38 mainly accumulated inFR-positive MDA-MB 231 tumor xenografts and kidneys. However,accumulation of the OTL38 in both organs was inhibited by theadministration of 100 fold excess of folic acid prior to injection ofOTL38 (FIGS. 17B and D) indicating that saturation of FR prevents thebinding of OTL38 to folate receptor. Therefore, these data suggest thatOTL38 is highly specific for FR.

(a) Administering to Tumor Xenografts (A549 Cells) that do not ExpressFR

Whole body imaging of A549 tumor xenograft model demonstrated that OTL38did not accumulated in the FR-negative tumors (FIG. 18A). Analysis oftissue biodistribution (FIG. 18B) demonstrated that, no fluorescence wasobserved in FR-negative tumors. As anticipated, kidney uptake wasobserved. Therefore, we can conclude that OTL38 is highly specific forFR and it will not passively accumulate in tumors that do not expressFR.

4. CONCLUSIONS

In vitro cell binding studies using human cervical cancers demonstratedthat OTL38 binds to FR with high affinity and specificity. In vivo wholebody fluorescence imaging using a human ovarian cancer cell line tumorxenograft models showed that OTL38 mainly accumulate in the FR-positivetumor xenografts and kidneys. Ex vivo tissue biodistribution of samemice that we subjected to whole body imaging confirmed that OTL38 bindsto FR-positive tumors and kidneys with minimal or no uptake in otherhealthy tissues resulting in excellent tumor-to-background ratios. Exvivo biodistribution data from orthotopic ovarian model demonstratedthat OTL38 mainly accumulated in the ovary with a human ovarian tumorbut not in the healthy ovary or other tissues. Administration of OTL38to either FR-negative tumor xenograft model or by blocking the FR inFR-positive tumor xenograft model further verified specify andselectivity of OTL38 to FR.

The most suitable route for administration will vary depending upon thedisease state to be treated, or the location of the suspected conditionor tumor to be diagnosed. For example, for treatment of inflammatoryconditions and various tumors, local administration, includingadministration by injection directly into the body part to be irradiatedby the excitation light (e.g., intracavitarily) provides the advantagethat the targeting construct (e.g., fluorescently tagged antibodies) canbe administered in a high concentration without risk of thecomplications that may accompany systemic administration thereof.

The compounds of the present disclosure as well as any additionaltargeting constructs used in diagnostic cocktails comprising thecompounds of the present disclosure are administered in a “effectiveamount” for diagnosis. An effective amount is the quantity of atargeting construct necessary to aid in direct visualization of anytarget tissue located in the body part under investigation in a subject.A “subject” as the term is used herein is contemplated to include anymammal, such as a domesticated pet, farm animal, or zoo animal, butpreferably is a human. Amounts effective for diagnostic use will, ofcourse, depend on the size and location of the body part to beinvestigated, the affinity of the targeting construct for the targettissue, the type of target tissue, as well as the route ofadministration. Local administration of the targeting construct willtypically require a smaller dosage than any mode of systemicadministration, although the local concentration of the targetingconstruct may, in some cases, be higher following local administrationthan can be achieved with safety upon systemic administration.

Since individual subjects may present a wide variation in severity ofsymptoms and each targeting construct has its unique diagnosticcharacteristics, including, affinity of the targeting construct for thetarget, rate of clearance of the targeting construct by bodilyprocesses, the properties of the fluorophore contained therein, and thelike, the skilled practitioner will weigh the factors and vary thedosages accordingly.

The compounds of the present disclosure as well as cocktails comprisingthese compounds can be formulated as a sterile injectable suspensionaccording to known methods using suitable dispersing or wetting agentsand suspending agents. The sterile injectable preparation may also be asterile injectable solution or suspension in a non-toxicparenterally-acceptable diluent or solvent, for example, as a solutionin 1-4, butanediol. Sterile, fixed oils are conventionally employed as asolvent or suspending medium. For this purpose any bland fixed oil maybe employed, including synthetic mono- or diglycerides, fatty acids(including oleic acid), naturally occurring vegetable oils like sesameoil, coconut oil, peanut oil, cottonseed oil, etc., or synthetic fattyvehicles like ethyl oleate, or the like. Buffers, preservatives,antioxidants, and the like, can be incorporated as required, or,alternatively, can comprise the formulation.

It will be apparent to those skilled in the art that various changes maybe made in the disclosure without departing from the spirit and scopethereof, and therefore, the disclosure encompasses embodiments inaddition to those specifically disclosed in the specification, but onlyas indicated in the appended claims.

The examples that follow are merely provided for the purpose ofillustrating particular embodiments of the disclosure and are notintended to be limiting to the scope of the appended claims. Asdiscussed herein, particular features of the disclosed compounds andmethods can be modified in various ways that are not necessary to theoperability or advantages they provide. For example, the compounds canincorporate a variety of amino acids and amino acid derivatives as wellas targeting ligands depending on the particular use for which thecompound will be employed. One of skill in the art will appreciate thatsuch modifications are encompassed within the scope of the appendedclaims.

1. A composition for use in diagnostic imaging wherein the compositioncomprises a compound having the formula:

or a pharmaceutically acceptable salt of said compound or isotopethereof, wherein: X is an single amino acid or a single amino acidderivative thereof, wherein the single amino acid or single amino acidderivative contains an —OH, —NH₂, or —SH functional group, and Y is adye that has a fluorescence excitation and emission spectra in the nearinfra-red range, wherein Y is represented by the formula:

wherein, R¹ is independently selected from the group consisting of O, S,N and C, and R² is independently selected from the group consisting ofCH₂ and CH₂CH₂.
 2. The composition of claim 1, wherein the single aminoacid is selected from the group consisting of tyrosine, cysteine,serine, and lysine.
 3. The composition of claim 2, wherein the singleamino acid of the compound is tyrosine.
 4. The composition of claim 1,wherein the single amino acid derivative is selected from the groupconsisting of a derivative of tyrosine, a derivative of cysteine, aderivative of serine and a derivative of lysine.